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Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis
Reference genes are essential for gene expression analysis when using real-time quantitative PCR (RT-qPCR). Xenopus laevis is a popular amphibian model for studying vertebrate embryogenesis and development. Further, X. laevis is ideal for studying thyroid signaling due to its thyroid dependent metam...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764961/ https://www.ncbi.nlm.nih.gov/pubmed/29323148 http://dx.doi.org/10.1038/s41598-017-18684-1 |
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author | Mughal, Bilal B. Leemans, Michelle Spirhanzlova, Petra Demeneix, Barbara Fini, Jean-Baptiste |
author_facet | Mughal, Bilal B. Leemans, Michelle Spirhanzlova, Petra Demeneix, Barbara Fini, Jean-Baptiste |
author_sort | Mughal, Bilal B. |
collection | PubMed |
description | Reference genes are essential for gene expression analysis when using real-time quantitative PCR (RT-qPCR). Xenopus laevis is a popular amphibian model for studying vertebrate embryogenesis and development. Further, X. laevis is ideal for studying thyroid signaling due to its thyroid dependent metamorphosis, a stage comparable to birth in humans. When using PCR based studies, a primary concern is the choice of reference genes. Commonly used references are eef1a1, odc1, rpl8, and actnB, although there is a lack of ad hoc reference genes for X. laevis. Here, we used previously published RNA-seq data on different X. laevis stages and identified the top 14 candidate genes with respect to their expression levels as a function of developmental stage and degree of variation. We further evaluated the stability of these and other candidate genes using RT-qPCR on various stages including the unfertilised eggs, whole embryos during early development and brains during late development. We used four different statistical software packages: deltaCT, geNorm, NormFinder and BestKeeper. We report optimized reference gene pair combinations for studying development (early whole embryos), brains at later stages (metamorphosis and adult), and thyroid signalling. These reference gene pairs are suitable for studying different aspects of X. laevis development and organogenesis. |
format | Online Article Text |
id | pubmed-5764961 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-57649612018-01-17 Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis Mughal, Bilal B. Leemans, Michelle Spirhanzlova, Petra Demeneix, Barbara Fini, Jean-Baptiste Sci Rep Article Reference genes are essential for gene expression analysis when using real-time quantitative PCR (RT-qPCR). Xenopus laevis is a popular amphibian model for studying vertebrate embryogenesis and development. Further, X. laevis is ideal for studying thyroid signaling due to its thyroid dependent metamorphosis, a stage comparable to birth in humans. When using PCR based studies, a primary concern is the choice of reference genes. Commonly used references are eef1a1, odc1, rpl8, and actnB, although there is a lack of ad hoc reference genes for X. laevis. Here, we used previously published RNA-seq data on different X. laevis stages and identified the top 14 candidate genes with respect to their expression levels as a function of developmental stage and degree of variation. We further evaluated the stability of these and other candidate genes using RT-qPCR on various stages including the unfertilised eggs, whole embryos during early development and brains during late development. We used four different statistical software packages: deltaCT, geNorm, NormFinder and BestKeeper. We report optimized reference gene pair combinations for studying development (early whole embryos), brains at later stages (metamorphosis and adult), and thyroid signalling. These reference gene pairs are suitable for studying different aspects of X. laevis development and organogenesis. Nature Publishing Group UK 2018-01-11 /pmc/articles/PMC5764961/ /pubmed/29323148 http://dx.doi.org/10.1038/s41598-017-18684-1 Text en © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Mughal, Bilal B. Leemans, Michelle Spirhanzlova, Petra Demeneix, Barbara Fini, Jean-Baptiste Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis |
title | Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis |
title_full | Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis |
title_fullStr | Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis |
title_full_unstemmed | Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis |
title_short | Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis |
title_sort | reference gene identification and validation for quantitative real-time pcr studies in developing xenopus laevis |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764961/ https://www.ncbi.nlm.nih.gov/pubmed/29323148 http://dx.doi.org/10.1038/s41598-017-18684-1 |
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